Our major goal is to understand how the polymerization of actin following platelet stimulation is regulated. Resting platelets contain 280 muM monomeric actin which, as we have recently discovered, is mostly bound to thymosin beta4. In preliminary studies we have found unidentified capping and cutting activities, both calcium insensitive, in supernatants of permeabilized platelets. We will purify and characterize the proteins responsible for these activities. We will measure the total amount of capper(s) and the Kd(s) to determine whether the actin filaments in resting platelets are capped. Fully capped filaments would indicate that the steady state free G-actin concentration is above barbed end critical, so that polymerization could be elicited solely by the generation of free barbed ends. These could be produced by uncapping: we will study whether the cutting protein generates free barbed ends. We have obtained preliminary evidence for nucleating sites at the platelet membrane and will characterize these sites further. Actin polymerization is very rapid: we will determine whether there is a role for profilin, in low as well as high affinity profilin-actin complexes, in increasing the speed of polymerization. Our second goal is to study whether shifting a significant fraction of polymerized actin into the monomer pool alters actin synthesis (i.e. up or down regulation of actin synthesis) and total actin in the cell. Preliminary evidence shows that transfection with a plasmid containing full length cDNA for Tbeta4 is followed by a striking reduction in stress fibers. We will study sustained effects of such transfections. For this purpose we will generate cell lines with increased levels of Tbeta4 using transfection with plasmid containing resistance markers and inducible promoters.